Unveiling the microevolution of antimicrobial resistance in selected Pseudomonas aeruginosa isolates from Egyptian healthcare settings: A genomic approach

The incidence of Pseudomonas aeruginosa infections in healthcare environments, particularly in low-and middle-income countries, is on the rise. The purpose of this study was to provide comprehensive genomic insights into thirteen P. aeruginosa isolates obtained from Egyptian healthcare settings. Phenotypic analysis of the antimicrobial resistance profile and biofilm formation were performed using minimum inhibitory concentration and microtiter plate assay, respectively. Whole genome sequencing was employed to identify sequence typing, resistome, virulome, and mobile genetic elements. Our findings indicate that 92.3% of the isolates were classified as extensively drug-resistant, with 53.85% of these demonstrating strong biofilm production capabilities. The predominant clone observed in the study was ST773, followed by ST235, both of which were associated with the O11 serotype. Core genome multi-locus sequence typing comparison of these clones with global isolates suggested their potential global expansion and adaptation. A significant portion of the isolates harbored Col plasmids and various MGEs, all of which were linked to antimicrobial resistance genes. Single nucleotide polymorphisms in different genes were associated with the development of antimicrobial resistance in these isolates. In conclusion, this pilot study underscores the prevalence of extensively drug-resistant P. aeruginosa isolates and emphasizes the role of horizontal gene transfer facilitated by a diverse array of mobile genetic elements within various clones. Furthermore, specific insertion sequences and mutations were found to be associated with antibiotic resistance.


MLST and serotyping
In silico analysis of multilocus sequence typing (MLST) using the Oxford scheme identified five sequence types (STs).The majority of isolates were attributed to ST773 (6/13) and ST235 (3/13), with two isolates classified as ST664, and sporadic occurrences of ST1822 and ST274 were noted (Fig. 1).

Single nucleotide polymorphisms in antimicrobial resistance determinants
Mutations in ampR gene, conferring resistance to aztreonam, along with the presence of Pseudomonas-derived cephalosporinase (bla PDC16/35 ) were prominently detected.Additionally, we identified single nucleotide polymorphisms (SNPs) that could potentially impact gene expression using the resistance gene identifier (RGI) (Table 1).

Core genome MLST characterization
The relatedness of P. aeruginosa isolates assigned to ST773 and ST235 with globally reported isolates was investigated using the cgMLST scheme.This analysis involved a selected set of 13 publicly available P. aeruginosa isolates of ST773 from diverse geographical locations worldwide, and it included both cgMLST and phylogenetic reconstruction (Table S6).The analysis revealed a major cluster comprising our Egyptian isolates (Fig. 3), which clustered with isolates from Saudi Arabia.This genetic relevance can be attributed to geographical proximity.Additionally, the Egyptian isolates also clustered with isolates from the USA and Hungary, suggesting potential global expansion and adaptation (Fig. 3).
Furthermore, cgMLST analysis was performed using a selected set of 32 publicly available P. aeruginosa isolates of ST235 reported worldwide (Table S7).The analysis identified 3 clonal clusters, Cluster 1 was the largest consisting of our Egyptian isolates grouping with isolates from Saudi Arabia, Netherlands, Australia, Pakistan, Germany, China, and Indonesia.The remaining isolates were considered unrelated to our isolates and assigned unique clonal clusters (Fig. 4).
P. aeruginosa isolates, comprising 3 Egyptian and 32 worldwide isolates from diverse geographic locations.Distances are based on the differences in the 3867 alleles in P. aeruginosa cgMLST, with a cluster distance threshold set at 25. Nodes are labeled by country of isolation, and colored based on the presence of carbapenem and beta-lactam resistance genes within the genome.

Virulome analysis
Thirteen extensively drug-resistant (XDR) clinical isolates exhibited cytotoxic virulence patterns, with the detection of 87 virulence genes (Table S5) using the Virulence Factor Database (VFDB), of which only 15 were www.nature.com/scientificreports/incorporated into Fig. 5.Among the genes encoding effector proteins that play a role in T3SS, exoT emerged as the most predominant virulence gene and was detected in all screened isolates.Among screened isolates, twelve isolates harbored exoY, ten carried exoU, and three contained exoS.Additionally, the phospholipase gene (plcH), exotoxin A gene (toxA), and elastase B gene (lasB) were detected in all P. aeruginosa isolates, except for the elastase A gene (lasA), which was found in only 12 isolates.The T6SS effector phospholipase (pldA), the effector gene in H1-T6SS (hcp1), and ClpV1 were present in all isolates; however, plcB and plcN were absent in all isolates.

Association between biofilm formation, clonal distribution, and AMR
We examined the correlation between biofilm formation (Table S8) and extensively drug-resistant (XDR) phenotypes among P. aeruginosa isolates (Fig. 5).More than half of the isolates (53.85%) exhibited strong biofilm production, particularly associated with ST235 and ST773.

Discussion
In this study, we explored the population structure, virulence factors, and genomic alterations contributing to AMR, encompassing the acquisition of AMR genes through horizontal gene transfer and chromosomal mutations.Our findings highlight that the majority of our isolates exhibited XDR, aligning with a prior study documenting heightened resistance levels in P. aeruginosa strains originating from Egyptian clinical settings 23 .This poses significant challenges for infection control measures within the Egyptian health care settings and highlights the crucial need for enhanced infection prevention strategies, surveillance, and monitoring to mitigate the risk of spread of these pathogens.Approximately 92.3% of our isolates demonstrated resistance to ticarcillin and ticarcillin-clavulanic acid, as well as to various antipseudomonal antibiotics including ciprofloxacin, gentamicin, cefepime, amikacin, ceftazidime, tobramycin, and piperacillin-tazobactam. Notably, the resistance patterns observed align with those reported in various observational studies on hospital-acquired P. aeruginosa in Egypt [23][24][25] .It underscores the challenge of treating infections caused by resistant P. aeruginosa isolates within the Egyptian healthcare settings.The concerning resistance rates against carbapenem antibiotics like imipenem and meropenem signify a potential obstacle to effective treatment, emphasizing the importance of genomic surveillance and antibiotic stewardship practices.All isolates were susceptible to colistin, the last resort for the treatment of carbapenem-resistant Gram-negative infections.This suggests that colistin remains an effective therapeutic choice for addressing P. aeruginosa infections in the Egyptian healthcare settings.
Using the proposed cgMLST scheme, we successfully linked our major reported clones (ST773 and ST235) to distant isolates reported worldwide.Although ST773 was not previously classified as a high-risk clone 34,35 , our study together with other recent studies 36,37 suggests that ST773 is likely to emerge as a global clone.Furthermore, our findings indicate the local expansion of the ST773 lineage and the presence of diverse AMR determinants, enhancing its ability to propagate in epidemic settings.A major progression of the worldwide hyper virulent high-risk clone ST235 was documented in our study and several other studies 27,38 .This clone is playing a crucial role in the global prevalence of carbapenemase-producing isolates, and it has previously been associated with the acquisition of horizontally acquired resistance determinants across diverse geographical and environmental settings worldwide 39 .
Despite the limited number of isolates tested, the majority exhibited resistance to various antibiotics.Interestingly, each sequence type (ST) demonstrated a distinct profile of ARGs.For instance, ST773 isolates exclusively harbored bla OXA-395, bla PAO , qnrVC1, rmtB, and aadA10; while ST235 exclusively harbored bla OXA-488 , aph3'XV, aph3'lb and aph6'Id, as well as bla PDC2 was only detected in both STs.This observation further supports the potential clonal dissemination of resistance determinants and propagation of resistance traits within a particular lineage that is induced, at least in part, by selective pressures of antibiotic use in the Egyptian healthcare settings.
Beside clonal dissemination of resistant clones, recent studies highlighted the role of MGEs, encompassing IS, transposons, integrases, phages, and integrative elements in the dissemination ARGs at the clinical settings 40,41 .In this study, different IS, including ISUnCu1, ISPA100, and IS6100 were detected in P. aeruginosa ST773 isolates, whereas the presence of ISPa7 was exclusively present in ST1822.ISUnCu1 was found within the attC sites of aadA1 40,42 ; IS6100 was identified in gram-negative bacteria such as Pseudomonas, Salmonella, Klebsiella, Acinetobacter and Enterobacter, showing a variety of ARGs 41 ; and in agreement with our results, ISPa7 has been reported on a upstream region from a bla VIM gene 43 .
Our findings highlighted that a substantial number of isolates harbored Col plasmids from diverse subtypes, such as Col(pHAD28), Col440I, Col440II, ColpVC, ColRNAI, Col8282, and Col (KPHS6) were observed.These Col replicon subtypes have been previously associated with various carbapenemase genes, correlating with the observed high resistance to carbapenems in our study 44 .Consequently, these findings have significant implications for infection control strategies and antibiotic management.The high prevalence of resistance among our screened isolates, and the presence of plasmids and MGEs would facilitate the acquisition of new antimicrobial resistance determinants.Therefore, there is an urgent need for robust infection control measures and the implementation strict protocols to prevent the spread of resistant strains within healthcare settings.
P. aeruginosa possesses sophisticated secretion systems that facilitate the delivery of virulence factors, including toxins, elastases, lipases, and proteases into hots cells 8 .Consequently, our virulome analysis revealed the presence of virulence genes, shedding light on its pathogenicity and antibiotic resistance profiles.P. aeruginosa utilizes the T3SS to deliver its effector toxins (exoT, exoY, exoS, and exoU) into the host cells.This mechanism facilitates colonization and aids evasion from the immune system 60 .Interestingly, exoT was the most prevalent gene, along with exoY (except in S79).Additionally, exoS and exoU were found to be mutually exclusive.
Furthermore, a recent study revealed that P. aeruginosa utilizes H1-, H2-, and H3-T6SS to transport its toxins into both eukaryotic and prokaryotic cells via effectors pldA, hcp1, and ClpV1 61 .Consistent with these findings, we detected these encoding genes in our isolates.Additionally, the T2SS of P. aeruginosa is crucial for the release of different effector proteins, which play a significant role in damaging host cells and contributing to the pathogenicity of P. aeruginosa 62 .In this study, we observed the presence of toxA, lasB, and plcH in all screened isolates, while lasA was found in 12 isolates.
In conclusion, our pilot study has provided several significant insights into the differential clonal distribution of resistant P. aeruginosa isolates.It highlights the pivotal role of horizontal gene transfer and MGEs in bacterial adaptation, particularly in response to the selective pressure induced by antibiotic overuse in clinical settings, especially in low-or middle-income countries.These findings collectively underscore the urgent need for enhanced surveillance and strategic interventions to curb the spread of XDR P. aeruginosa in healthcare settings, given the complexity and adaptability of these bacterial strains.Further research and ongoing monitoring are imperative for a comprehensive understanding of the mechanisms driving antimicrobial resistance and the development of effective control strategies.

Bacterial isolation and identification
Thirteen non-duplicate historical P. aeruginosa isolates were collected from one microbiology laboratory serving as a diagnostic facility for multiple hospitals in Alexandria, Egypt, between August 2020 and March 2021, as previously mentioned 63 .Patient names remained anonymous.
Antibiotic resistance patterns are categorized into three main classes: MDR (Multi-Drug Resistance), characterized by resistance to at least one drug in three antibiotic classes; XDR, indicating resistance to most antibiotics except one or two classes; and PDR (Pan-Drug Resistance), indicating resistance to all available antibiotic agents 64 .

In vitro biofilm screening
Biofilm screening among tested isolates was conducted as previously described 67,68 with some modifications.Briefly, 20 µl of each bacterial isolate was added to 180 µl of fresh brain heart infusion broth (BHB) in a 96-well plate and incubated for 24 h at 37 °C.Subsequently, the wells were washed with 1X phosphate-buffered saline

Annotation and pangenome analysis
Contigs were annotated using Prokka 78 , and the GFF3 output files from Prokka were utilized for the pangenome analysis of the isolates.The Roary software 79 was employed to derive pangenome statistics for core, shell, and cloud genes.

In silico identification of resistome and virulome
Genome resistomes were analyzed using ABRicate (https:// github.com/ tseem ann/ abric ate) with ResFinder and Comprehensive Antibiotic Resistance Database (CARD) databases 80,81 .Removal of redundant genes from both databases was performed.Resistance genes were categorized based on the antibiotic classes.Additionally, chromosomal mutations and SNPs for AMR in each clinical isolate were identified by extracting the sequences of the selected resistance genes and running CARD resistance gene identifier (RGI) 81 .The virulome profile of the isolates was annotated based on VFDB in ABRicate.Virulence genes were organized according to virulence factors and phenotypes.

Phylogenetic tree construction
cgMLST was performed by fast-GeP.Core genome polymorphic genes (genes with at least one nucleotide difference among all genomes) were selected and concatenated by the ruby script concat_cgMLST_genes.rb (https:// github.com/ JoseC oboDi az/ concat_ cgMLST_ genes).The concatenated gene-by-gene fasta file was used for alignment and phylogenetic tree building using MAFFT version 7 (using default parameters for alignment and the Neighbor-Joining method, the Jukes-Cantor substitution model and 1000 bootstrap resampling for the construction of the phylogenetic tree) 82 .The cgMLST tree was visualized using iTOL (https:// itol.embl.de/) 83 .

Core genome MLST global comparison of predominant clones
The cgMLST comparison of ST773 and ST235 with globally reported isolates was performed using a well-defined scheme available in Ridom SeqSphere + v.8.3.5 software (Ridom GmbH, Münster, Germany), according to the P. aeruginosa sensu lato cgMLST' version 1.0 scheme https:// www.cgmlst.org/ ncs/ schema/ schema/ Paeru ginos a783/ which included 3867 genes of the P. aeruginosa core genome (cgMLST).Seqsphere + tool mapped the reads against the reference genome using BWA v 0. www.nature.com/scientificreports/Phred value > 30) and defined the cgMLST gene alleles.A combination of all these alleles in each isolate formed an allelic profile that was utilized to create a minimum spanning tree (MST) using Ridom SeqSphere + with the 'pairwise ignore missing values; % column difference' parameter.A threshold was set at ≤ 15 allelic differences paired with a cluster alert quality threshold of at least 85% good cgMLST targets to define the clusters.

Figure 1 .
Figure 1.Phylogenetic tree illustrating the distribution of antimicrobial resistance genes, serotypes and plasmids replicon among different STs.Inc types found within all the isolates are represented.Cerulean, pink, maroon, and navy colors represent the existence of genes/replicon plasmids; white indicates the absence of gene/ replicon plasmids.The figure was created using iTOL.

Figure 2 .
Figure 2. (a) Distribution of MGEs, including insertion sequences carrying different antimicrobial and antiseptic resistance genes were ST773.(b) Specific insertion sequences carrying ARGs found in ST1822.Both figures were created using ggplot2 and ggenes R packages.

Figure 3 .Figure 4 .
Figure 3. Minimum spanning tree (MST) of the 19 ST773 P. aeruginosa isolates (6 Egyptian and 13 worldwide isolates) collected from different geographical locations.Distances are based on the differences in the 3867 alleles in P. aeruginosa cgMLST, with a cluster distance threshold set at 25. Nodes are labeled by country of isolation, and colored based on the presence of carbapenem and beta-lactam resistance genes.

Figure 5 .
Figure 5. Presence of selected virulence genes and its correlation in the formation of biofilm.Maroon color showed the presence of these genes and white outlines the gene absence.Figure was created by iTOL.

Table 1 .
Mutations associated with antibiotic resistance.